Glycerol Detection 500 point kit (LIP
Contents
1. 25 12 5 6 25 3 125 uM uM uM uM Note The above dilution series generates enough volume to perform the standard curve in duplicate If you wish to perform the standard curve in duplicate please note that eight fewer data points can be assayed with this kit 8 At this time prepare the Glycerol Reagent A by adding 40 ml or 11 ml room temperature deionized water per bottle following the instructions on the bottle Gently invert bottle to mix contents DO NOT VORTEX Use a pipet to ensure that the powder is completely dissolved Store in a light protected bottle Reconstituted Glycerol Reagent A is stable for 60 days refrigerated 2 8 C store any remaining solution refrigerated 2 8 C You may order additional glycerol standards for the Lipolysis Kit cat LIP GLYSTAN 9 At the end of the incubation 100 ul of the conditioned media is removed and transferred to the corresponding well of another blank plate This is most easily accomplished using a multi channel pipet Add 100 ul of each glycerol standard to any remaining empty wells in one of the blank assay plates 10 Add the reconstituted Glycerol Reagent A solution to a disposable tray not provided Add 100 ul of Reagent A to each well of the assay plates containing samples Gently pipet up and down once to mix Pop the bubbles using a large gauge needle or a clean pipet tip The plate is then incubated at 25 C room temperature for 15 minutes 11 The optical densi2. 181 201 7 Rieusset J Chambrier C Bouzakri K Dussere E Auwerx J Riou J P Laville M Vidal H Diabetologia 2001 44 544 554 8 Robidoux J Martin TL Collins S 2004 Ann Rev Chem 253 7570 7578 9 Scriba D Aprath Husmann Blum WF Hauner H Eur J Endocrinol 2000 143 439 445 10 Snyder PB Emerging Therapeutic Targets 1999 3 4 587 599 11 Tansey JT Sztalryd C Hlavin EM Kimmel AR Londos C 2004 UBMB Life 56 7 379 85 Rev 06 2009 Page 9 of 11 APPENDIX A PLATE LAYOUT Rev 06 2009 Page 10 of 11 APPENDIX B PROCEDURE FLOWCHART Make all test compound dilutions in Assay Buffer Remove 120 ul media from all wells Add 200 ul Wash Buffer to all wells Remove 200 ul media amp Wash Buffer Add another 200 ul Wash Buffer to all wells Remove all media amp Wash Buffer Add 150 ul treatments controls to 3 wells at a time OPTION Add 100 ul well compounds to a fresh plate without cells Incubate 3 5 hours at 37 C One hour prior to assay reconstitute Glycerol Reagent A and prepare standards Keep all at room temp Remove 100 ul well conditioned media to a blank assay plate not provided Add standard dilutions Add 100 ul well reconstituted Glycerol Reagent A to the plate including the glycerol standards at 100ul well and optional plate without cells 4 Incubate at 25 C room temperature for 15 minutes Pop the bubbles in each well Measure the optical density o
3. Glycerol released to the medium is phosphorylated by adenosine triphosphate ATP forming glycerol 1 phosphate G 1 P and adenosine 5 diphosphate ADP in the reaction catalyzed by glycerol kinase G 1 P is then oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate DAP and hydrogen peroxide H202 A quinoneimine dye is produced by the peroxidase catalyzed coupling of 4 aminoantipyrine 4 AAP and sodium N ethyl N 3 sulfopropyl m anisidine ESPA with H202 which shows an absorbance maximum at 540nm The increase in absorbance at 540nm is directly proportional to glycerol concentration of the sample GLYCEROL ATP gt G 1 P ADP G 1 P O02 gt DAP H20 H20 4 AAP ESPA gt Quinoneimine dye H20 Rev 06 2009 Page 3 of 11 ITEMS INCLUDED IN THE KIT ITEM DESCRIPTION UNIT QTY STORAGE LIP 1 Assay Buffer 500m ml BOTTLE Wash Buffer 250ml a kazaa KC Reagent A 40 C E Reconstitute with 40 ml can cat RGTA 40 deionized water prior to use BOTTLE Glycerol Reagent A 11 ml Reconstitute with 11 ml 11ML cat RGTA 10 deionized water prior to use some Glycerol standard Glycerol 1mM Reconstitute 100 ul Ea Orange cap make the 200 uM glycerol standard see page 6 for recommended dilution scheme Vehicle Control 0 1 DMSO in Assay Buffer 1ml 20 C kiki Positive control Isoproterenol 10 mM in 10 ul 2 20 C Blue cap DMSO Dilute to 1 uM_in VIAL A
4. er dose of the compound at treatment OR a dilute solution of the condition medium at the time of the assay The R value should be equal or greater then 0 98 for the standard curve to be valid Any R values below 0 98 must have the standard curve run again Data are expressed as uM glycerol released OPTION express data as Fold induction over appropriate vehicle Fold induction uM glycerol SAMPLE uM glycerol VEHICLE Rev 06 2009 Page 7 of 11 TROUBLESHOOTING Problem Suggestions High background or the glycerol e Change pipet tips frequently reagent A turns purple before the e Use Glycerol Reagent A before the expiration date assay begins No response to positive control eDo not add the compounds and controls too fast The cells can float if a solution is added too fast e Make sure to starve the cells for 5 7 days BEFORE initiating treatment Edge effects e Ensure a saturated humidity in the incubator to prevent evaporation from the outside wells Inconsistent OD reading e The Assay Buffer contains bovine serum albumin BSA Be careful when pipetting to avoid bubbles If bubbles persist burst the bubbles using a large gauge needle and read the plate again FREQUENTLY ASKED QUESTIONS 1 want to perform a lipolysis time course experiment How many time points can complete We do not recommend performing more than 2 time points per assay For time course experiments add 250 ul assay mediu
5. f each well at 540 nm using a spectrophotometer plate reader Rev 06 2009 Page 11 of 11 O00000000000 O00000000000 O00000000000 O00000000000 000000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 000000000000 O00000000000 O00000000000 O00000000000 000000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 0 010000 010 0 0 0 0 070 00 0 0 01010 0 0 0 070 00 0 0 01010 010 0 070 00 0 0 01010 010 0 070 00 00 01010 010 0 120 ul media 200 ul Wash Buffer 200 ul Wash Buffer Add another 200 ul Wash Buffer Remove 3 wells at a time Add treatments 3 wells at a time 0 0 0700 0 010 0 010 0 100 yl 200000000000 OO gt 000000000000 GLYCEROL REAGENT A 0 00 0 00 0 00 0 0 0 0 00 An additional blank assay plate may be necessary for the assay of glycerol standards if al 96 wells are used not provided
6. gy balance Lipolysis is the process in which triglycerides TG are hydrolyzed into glycerol and free fatty acids This process releases free fatty acids FFA into the bloodstream where they may be either re esterified by the adipocyte or travel to other tissues and exert other effects throughout the body Elevated adipocyte lipolysis has been observed in obese and diabetic individuals Arner 1996 Excessive free fatty acid production is believed to contribute to insulin resistance in skeletal muscle that is observed in obesity Hormone sensitive lipase is the rate limiting enzyme catalyzing triglyceride breakdown Perilipins one of the PAT perilipins adipophilin TIP47 proteins family of lipid associated proteins are implicated in adipocyte lipolysis by mediating the interaction of HSL with the triacylglycerol molecule Brasaemle et al 2004 reviewed in Tansey et al 2004 The presence of these proteins corresponds to lipolytic stimulation in cultured adipocytes Braemle et al 2004 The sympathetic nervous system also plays a key role in the regulation of lipid mobilization The main lipolytic pathway involves beta agonists fB agonists which activate B adrenergic receptors via the intracellular G proteins in adipocytes This leads to the activation of adenylate cyclase AC which then increases cyclic AMP cAMP levels Elevated cAMP acts as a second messenger to activate hormone sensitive lipase HSL HSL the rate limiting enzyme regu
7. isoproterenol in assay buffer as the positive control not provided in this kit Use the Assay Buffer alone as one of the vehicle controls Please be sure to include your vehicle if your test compounds are not dissolved in DMSO The assay should be performed in triplicate 5 OPTION to determine if the compound alone reacts with the Glycerol Reagent A prepare a fresh plate not included in kit containing 100 ul of the compound This plate can be incubated at 37 C with the treated cells When performing the assay add 100 ul of Glycerol Reagent A following the instructions in Steps 10 and 11 6 Incubate the plates at 37 C humidified incubator for 3 hours for time course experiments the longest time point is usually 24 hours 7 One hour prior to the assay prepare the glycerol standards as follows Briefly spin down the contents of the glycerol standard tube before reconstitution Pipette 400 ul of Wash Buffer into the 1 mM glycerol standard tube provided and mix well by vortexing This produces a diluted stock glycerol standard of 200 uM Pipette 250 ul of wash buffer into 6 tubes not provided Using the newly diluted stock glycerol solution prepare a dilution series as depicted below Mix each new dilution Rev 06 2009 Page 5 of 11 thoroughly before proceeding to the next The 200 uM stock dilution serves as the highest standard and the wash buffer serves as the zero standard 400 pl 250 pl 250 pl 250 pl 250 pl 250 pl 250 pl
8. lating adipocyte lipolysis then catalyzes the hydrolysis of triglycerides and results in the release of glycerol and FFA increased lipolysis Phosphodiesterases PDE are enzymes that hydrolyze cAMP to 5 AMP 5 prime adenosine monophosphate This action results in a decrease in lipolysis PDE inhibitors increase intracellular cAMP levels 3 isobutyl 1 methylxanthine IBMX a non specific inhibitor of cAMP phosphodiesterases PDE is used as the positive control if your test compounds are suspected PDE inhibitors Isoproterenol a non specific B adrenergic agonist is used as the positive control if your test compounds affect lipolysis via B adrenergic receptors Robidoux et al 2004 This lipolysis assay kit provides the tool to study chemical compounds that may influence lipolysis in cultured human adipocytes This kit detects lipolysis as the amount of glycerol released from mammalian cells in a 96 well format This kit contains sufficient volume of reagents to assay 500 data points 5 96 well plates Rev 06 2009 Page 2 of 11 Figure 1 Overview of adipocyte lipolysis haa _ ABBREVIATIONS AC AR G i Foo 77 HSE Z ee te TG FFA g lycerol FFA glycerol lt gt bloodstream PRINCIPLE OF THE ASSAY adenylate cyclase adrenergic receptors G protein coupled receptor free fatty acids protein kinase adenosine monophosphate adenosine triphosphate insulin receptor phosphodiesterase triglyceride
9. m with treatments per well Remove 100 ul for each time point Complete the assay using an equal volume Glycerol Reagent A 2 do not have time to perform the assay Can I freeze the conditioned media How long can store the samples before complete the assay Yes The conditioned media can be immediately stored at 80 C for a maximum of 7 days in regular polystyrene cell culture plates Bring the conditioned media in the plate to room temperature BEFORE adding the Glycerol Reagent A and completing the assay 3 Can buy the reagents separately The Glycerol Standard cat LIP GLYSTAN and Glycerol Reagent A cat RGTA 10 11 ml RGTA 40 40 ml are sold separately Assay Buffer is not sold separately 4 need to know the concentration of the BSA in the Assay Buffer ZenBio Inc does not provide the concentrations of the components of our media and buffers If knowledge of the BSA concentration is critical to your experiment you may order Assay Buffer WITHOUT BSA for no additional charge Please note it on your order Rev 06 2009 Page 8 of 11 REFERENCES 1 Arner P 1996 Diabetes Rev 4 4 450 463 2 Botion LM amp Green A Diabetes 1999 48 1691 1697 3 Brasaemle DL Dolios G Shapiro L Wang R 2004 J Biol Chem 279 45 46835 42 4 Cooper DMF Schlegel W Lin MC Rodbell M 1979 J Biol Chem 254 18 8927 8931 5 Dyck DJ Can J Appl Physiol 2000 25 6 495 523 6 Kordik CP amp Reitz AB J Medicinal Chem 1999 42 2
10. ssay Buffer before use i e 1 ulin 10 ml Assay Buffer Other equipment reagents required but not provided with the kit e Clean disposable pipettor trays Blank 96 well plates Multi channel Pipet single channel pipet and pipet tips Plate reader with a filter of 540 nm Incubator at 37 C Large gauge needle Cultured human adipocytes Tubes for diluting glycerol standards Rev 06 2009 Page 4 of 11 ASSAY PROCEDURE 1 Please observe your cells under a microscope prior to performing the assay 2 Make your stock solution using whatever vehicle is appropriate for your test compounds Dilute your stock solutions to their final concentration in Assay Buffer 500 ml is available NOTE if desired maintain a constant concentration of solvent by preparing all compound dilutions in the highest concentration of that solvent Dilute your controls in Assay Buffer Prepare all vehicles as appropriate for your compounds Include the Assay Buffer alone as a vehicle control PLEASE NOTE ZEN BIO DOES NOT RECOMMEND THE USE OF SOLVENTS AT CONCENTRATIONS ABOVE 1 3 Remove 120 ul medium from each well Gently add 200 ul Wash Buffer to all wells Remove 200 ul of the media and Wash Buffer from each well and replace with another 200 ul Wash Buffer 4 Remove all the media and Wash Buffer from the cells from triplicate wells Treat the cells with 150 ul of the test compounds resuspended in Assay Buffer three 3 wells at a time You can treat with
11. ty of each well is then measured at 540 nm Rev 06 2009 Page 6 of 11 GLYCEROL STANDARD CURVE Generate standard curve see example below DO NOT use this standard curve to generate your data This is an example Subtract the OD value of the OuM standard from all OD values including the standard curve uM OD OD ae Glycerol Standard Curve glycerol OD OD blank blank blank a 0 0 044 0 041 0 043 0 600 3 125 0 054 0 053 0 012 0 011 0 011 Ga 6 25 0 062 0 063 0 020 0 021 0 020 12 5 0 083 0 084 0 041 0 042 0 041 25 0 126 0 125 0 084 0 083 0 083 0 300 Linear Seriesi 50 0 205 0 208 0 163 0 166 0 164 100 0 372 0 374 0 330 0 332 0 331 200 0 698 0 697 0 656 0 655 0 655 0 000 0 50 100 150 200 250 Slope 0 003 uM Glycerol Intercept 0 001 R 1 000 y observed O D minus the blank x concentration of glycerol in uM To calculate x for each y i e to change the observed O D into glycerol concentration use the following equation y slope times x plus intercept y mx b so x y b m x y 0 001 0 003 where 0 003 slope of the line and 0 001 y intercept Be careful to enter the proper sign for the y intercept value as it may be a negative number Any OD values greater than the highest standard 200 uM should be suspect The compound should be re assayed using a low
12. zenbio gt Cultured Human Adipocyte Lipolysis Assay Kit 500 point assay kit 96 well Format Cat LIP 1 RB INSTRUCTION MANUAL 2ZBM0027 04 STORAGE CONDITIONS All orders are delivered via Federal Express Priority courier at ambient temperature All orders must be processed immediately upon arrival Glycerol Reagent A amp Buffers Store at 4 C Glycerol Standards amp Controls Store at 20 C ALL ZEN BIO INC PRODUCTS ARE FOR RESEARCH USE ONLY NOT APPROVED FOR HUMAN OR VETERINARY USE OR FOR USE IN DIAGNOSTIC OR CLINICAL PROCEDURES LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product No other warranties of any kind expressed or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Zen Bio Inc Zen Bio Inc shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product ORDERING INFORMATION AND TECHNICAL SERVICES Rev 06 2009 Zen Bio Inc 3200 Chapel Hill Nelson Blvd Suite 104 PO Box 13888 Research Triangle Park NC 27709 Telephone 919 547 0692 Facsimile FAX 919 547 0693 Toll Free 1 866 ADIPOSE 866 234 7673 Electronic mail e mail information zen bio com World Wide Web http www zenbio com Page 1 of 11 INTRODUCTION Lipolysis plays a central role in the regulation of ener
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